UTR Luciferase Reporter Research Articles

MiR-1908 Overexpression Inhibits Proliferation, Changing Akt Activity and p53 Expression in Hypoxic NSCLC Cells.

The ribosomal protein (RP)–p53 pathway has been shown to play a key role in apoptosis and senescence of cancer cells. miR-1908 is a newly found miRNA that was reported to have prognostic potential in melanoma. However, its role and mechanism in the progression of non-small cell lung cancer (NSCLC) are largely unknown. In this study, we found that expression of miR-1908 was significantly downregulated in human NSCLC cell lines, including SK-MES-1, A549, and NCI-H460. Then the role of miR-1908 in NSCLC cell proliferation was explored. The miR-1908 mimic was transfected into NSCLC cell lines, and their proliferation was detected. MTT and Cell Titer-Blue H analyses showed that the cell proliferation was notably reduced by the miR-1908 mimic transfection. Moreover, we found the RP–p53 pathway was activated by miR-1908 mimic. Moreover, the miR-1908 inhibitor transfection had a completely opposite effect on the NSCLC cell proliferation than that of miR-1908 mimic. To explore the underlying mechanism of that, TargetScan bioinformatics server and 3′-UTR luciferase reporter assay were applied to identify the targets of miR-1908. Our results showed that AKT1 substrate 1 (AKT1S1), a newly proven suppressor of the RP–p53 pathway, was a target of miR-1908, suggesting a probable mechanism for miR-191 suppressing NSCLC cell proliferation. Our findings provide a novel molecular target for the regulation of NSCLC cell proliferation.

MicroRNA-124 negatively regulates LPS-induced TNF-α production in mouse macrophages by decreasing protein stability.

MicroRNAs play pivotal roles in regulation of both innate and adaptive immune responses. In the present study, we investigated the effects of microRNA-124 (miR-124) on production of the pro-inflammatory cytokine TNF-α in lipopolysaccharide (LPS)-treated mouse macrophages. Mouse macrophage cell line RAW264.7 was stimulated with LPS (100 ng/mL). The levels of miR-124 and TNF-α mRNA were evaluated using q-PCR. ELISA and Western blotting were used to detect TNF-α protein level in cell supernatants and cells, respectively. 3'-UTR luciferase reporter assays were used to analyze the targets of miR-124. For in vivo experiments, mice were injected with LPS (30 mg/kg, ip). LPS stimulation significantly increased the mRNA level of miR-124 in RAW264.7 macrophages in vitro and mice in vivo. In RAW264.7 macrophages, knockdown of miR-124 with miR-124 inhibitor dose-dependently increased LPS-stimulated production of TNF-α protein and prolonged the half-life of TNF-α protein, but did not change TNF-α mRNA levels, whereas overexpression of miR-124 with miR-124 mimic produced the opposite effects. Furthermore, miR-124 was found to directly target two components of deubiquitinating enzymes: ubiquitin-specific proteases (USP) 2 and 14. Knockdown of USP2 or USP14 accelerated protein degradation of TNF-α, and abolished the effect of miR-124 on TNF-α protein stability. miR-124, targeting USP2 and USP14, negatively regulates LPS-induced TNF-α production in mouse macrophages, suggesting miR-124 as a new therapeutic target in inflammation-related diseases.

Abstract A27: Zeb1 induces LOXL2-mediated collagen stabilization and deposition in the extracellular matrix to drive lung cancer invasion and metastasis

Abstract Introduction: Lung cancer is the leading cause of cancer-related death, primarily due to distant metastatic disease. Metastatic cancer cells undergo an epithelial-to-mesenchymal transition (EMT) regulated by a double-negative feedback loop between the microRNA-200 (miR-200) family and Zeb1, but the precise mechanisms of Zeb1-dependent EMT in promoting malignancy remain largely undefined. While the cell-intrinsic effects of EMT are important for tumor progression, the reciprocal dynamic crosstalk between mesenchymal cancer cells and the extracellular matrix (ECM) is equally critical in regulating invasion and metastasis. This study investigates the collaborative effect of EMT and ECM in the metastatic process. Methods: Bioinformatic analysis of TCGA dataset was done to correlate ECM-associated gene expression with EMT gene signature scores. Western blotting and qPCR analysis of epithelial and mesenchymal lung cancer cell lines were performed to determine expression levels of collagen, LOX, and LOXL2. Lung tumor tissues from non-metastatic KrasG12D and metastatic KrasG12D;p53R172H mutant mice were analyzed by immunohistochemistry, Masson's trichrome staining, and second harmonics generation for collagen, LOX, and LOXL2 expression as well as collagen fiber organization. Syngeneic primary tumors generated by subcutaneous injection of murine lung cancer cell lines were analyzed in a similar fashion. Promoter and 3′-UTR luciferase reporter assays were performed to determine direct regulation LOXL2 and LOX by Zeb1 and miR-200, respectively. Results: Our results reveal increased collagen deposition in metastatic tumor tissues as a direct consequence of amplified collagen gene expression in Zeb1-activated mesenchymal lung cancer cells. Additionally, collagen fibers in metastatic lung tumors exhibit greater linearity and organization as a result of collagen crosslinking by the lysyl oxidase (LOX) family of enzymes. Expression of the LOX and LOXL2 isoforms is directly regulated by miR-200 and Zeb1, respectively, and their upregulation in metastatic tumors and mesenchymal cell lines is coordinated to that of collagen. Functionally, LOXL2, as opposed to LOX, is the principle isoform driving lung cancer metastasis by crosslinking and stabilizing insoluble collagen deposition in primary tumor tissues. Conclusions: Our study demonstrates that mesenchymal lung cancer cells metastasize by modulating the compositional and structural properties of the ECM through LOXL-mediated collagen crosslinking and deposition. We are the first to validate direct regulation of LOX and LOXL2 by the miR-200/Zeb1 axis, delineate collagen as a prognostic marker for lung cancer, and identify LOXL2 as a potential therapeutic target against tumor progression. Citation Format: David H. Peng, Pan Tong, Lauren A. Byers, Jing Wang, Chad J. Creighton, Don L. Gibbons. Zeb1 induces LOXL2-mediated collagen stabilization and deposition in the extracellular matrix to drive lung cancer invasion and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A27.

Mesenchymal Stem Cells Alleviate LPS-Induced Acute Lung Injury in Mice by MiR-142a-5p-Controlled Pulmonary Endothelial Cell Autophagy

Background/Aims: Damages of pulmonary endothelial cells (PECs) represent a critical pathological process during acute lung injury (ALI), and precede pulmonary epithelial cell injury, and long-term lung dysfunction. Transplantation of mesenchymal stem cells (MSCs) has proven therapeutic effects on ALI, whereas the underlying mechanisms remain ill-defined. Method: We transplanted MSCs in mice and then induced ALI using Lipopolysaccharides (LPS). We analyzed the changes in permeability index and lung histology. Mouse PECs were isolated by flow cytometry based on CD31 expression and then analyzed for autophagy-associated factors LC3 and Beclin-1 by Western blot. Beclin-1 mRNA was determined by RT-qPCR. In vitro, we performed bioinformatics analyses to identify the MSCs-regulated miRNAs that target Beclin-1, and confirmed that the binding was functional by 3'-UTR luciferase reporter assay. Results: We found that MSCs transplantation significantly reduced the severity of LPS-induced ALI in mice. MSCs increased autophagy of PECs to promote PEC survival. MSCs increased Beclin-1 protein but not mRNA. MiR-142a-5p was found to target the 3'-UTR of Beclin-1 mRNA to inhibit its protein translation in PECs. MSCs reduced the levels of miR-142a-5p in PECs from LPS-treated mice. Conclusion: MSCs may alleviate LPS-ALI through downregulation of miR-142a-5p, which allows PECs to increase Beclin-1-mediated cell autophagy.

Histone Deacetylase Inhibitors Induce microRNAs Targeting BTK in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a highly heterogeneous disease primarily affecting the elderly. Despite considerable progress in understanding the biology of AML, the prognosis of the majority of patients still remains poor due to lack of progress in improving therapy over the past 3 decades, thus creating the need to identify new pharmacological strategies.Bruton's Tyrosine Kinase (BTK) has been demonstrated to be functionally important in a variety of hematological malignancies and several groups have recently shown that BTK is highly expressed and constitutively active in AML cell lines and in a subset of AML patients. Moreover, treatment with ibrutinib, a covalent irreversible inhibitor of BTK, can reduce proliferation, blast adhesion to bone marrow stromal cells and migration of both AML cell lines and primary AML blasts. Validation of this target also occurred with shRNA experiments confirming the specificity of BTK for growth inhibition of AML.Dysregulation of microRNA (miRNA) expression contributes to the pathogenesis of numerous human malignancies including AML, where multiple miRNAs have been associated with specific cytogenetic and molecular subsets of the disease.Using a target prediction algorithm we identified several miRNAs as potential regulators of BTK (miR-147b, miR-210-3p, miR-425-5p, miR-1253, miR-4269, miR-4667-3p). We performed 3′UTR luciferase reporter assays in the AML cell line OCI-AML3 and found that relative luciferase activities were significantly decreased (20-30%) in cells transfected with miR-147b, miR-425-5p, miR-4269 and miR-4667-3p mimics. Flow cytometry and western blotting analysis confirmed that BTK protein expression was significantly down-regulated by all the predicted miRs except miR-1253.Previous data from our lab have shown that histone deacetylase (HDAC) 1 and 2 become recruited to multiple microRNA promoters, including the ones targeting BTK. To determine the consequence of HDAC inhibition on BTK signaling, we exposed primary blasts from 14 AML patients to 10nM of the pan-deacetylase inhibitor panobinostat (LBH589) for 48h. Our results showed that panobinostat induced a 2-23 fold increase in the expression miR-147b, miR-210-3p, miR-425-5p, and miR-4667-3p in most of the patients and a time-dependent depletion of total BTK protein and downstream BTK signaling (phospho- and total ERK and phospho- and total AKT) in about half of them. BTK protein at 24h directly correlated with BTK and HDAC1 mRNA expression at 24h and miR-210 induction at 24h inversely correlated with BTK mRNA levels at 24h. Moreover, 2 days of incubation with 10nM panobinostat is able to induce robust cell death in AML primary samples (9.6-92% apoptosis).We hypothesized that HDAC inhibitors would cooperate with kinase inhibitors of BTK to synergistically abolish BTK signaling and induce death in AML cells. Therefore, we treated OCI-AML3 and MV4-11 cell lines with 0.40 µM abexinostat, 1 µM ibrutinib for 2h alone followed by washout, or a combination of 0.40 µM abexinostat with 1 µM ibrutinib for 2h, and viability was measured at 48h. The combination of abexinostat and ibrutinib induced greater than additive cytotoxicity compared to either abexinostat or ibrutinib alone. Exposure to ibrutinib alone for 2h followed by washout inhibited the kinase activity of BTK (measured by the auto-phosphorylation on Y223) but did not affect total BTK protein. However, the combination of abexinostat and ibrutinib showed both loss of BTK kinase and decrease in total BTK protein, in addition to attenuation of BTK signaling, thus confirming the dual targeting of BTK via independent mechanisms.Our results provide the rationale for the clinical evaluation of ibrutininb in combination with histone deacetylase inhibitors in AML patients. DisclosuresNo relevant conflicts of interest to declare.

Microenvironment Regulation of IL23R/IL-23 Axis Drives Chronic Lymphocytic Leukemia (CLL) Progression

Background: CLL displays a considerable degree of clinical heterogeneity, which is in part ascribable to clone-intrinsic biological features and that are also influenced by clone-extrinsic events related to the microenvironment. Among the dynamics-taking place within the CLL microenvironment, those finalized to the induction of an overly inflammatory milieu may significantly impact on the CLL natural history by hijacking the immunological microenvironment at the same time fostering clone fitness. IL-23 acts as a prototypical pro-inflammatory mediator representing a promising therapeutic target. We analyzed the ability of CLL cells to sense IL-23 through the IL-23R complex (consisting of IL12Rß1 and IL23R subunits) expression and correlated this feature with clinical outcome. Moreover, we investigated the synthesis of IL-23 within the CLL microenvironment, and tested the biological effects of the IL-23/IL-23R axis engagement and of its interference in vitro and in vivo.Methods: IL23R complex was detected by quadruple flow cytometry staining with CD19, CD5, IL23R, and IL12Rβ1 in prospectively enrolled CLL cases (O-CLL1 protocol, clinicaltrial.gov identifier NCT00917540). On human tissue specimens, lymph node and bone marrow samples from 16 CLL patients were selected for in situ immunolocalization analyses. NOD/Shi-scid/γcnull (NSG) mice were used for in vivo xenografts, in which activated autologous T cells (AAT), obtained by adding anti-CD3 and CD28 Dynabeads and rIL2 were co-injected with CLL cells. MiRNA analysis was performed by Agilent's Human V2 platform and by quantitative PCR. MirVANA microRNA mimics and inhibitors were purchased from Ambion, Inc. For 3'UTR luciferase reporter experiments, miRNA target reporter vectors were purchased from Origene.Results: By flow cytometry, circulating CLL cells of 281 cases variably expressed IL23R side chain while consistently lacking IL12Rß1 chain expression. The engagement of the uncoupled IL23R complex expression (i.e. IL23R but not IL12Rb1 expression) by IL23 did not activate downstream signaling pathways, such as the up-regulation of pSTAT3. The 3-year TTFT probability of patients with low IL23R expression (IL23R-low) was 91% as compared to 75% of IL23R-high cases [χ2 9.1, P=.003; HR=3.2, 95%CI (1.4-7.1)]; in a multivariate model, IL23R expression still remained independently associated with TTFT. We explored the potential control of IL23R expression in CLL cells by miRNA and found 15 miRNAs inversely associated with IL23R expression, five of which predicted as regulators (miRNA-146b-5p, miRNA-155, miRNA-324-5p, miRNA-532-3p and miRNA-630). Among these, miR-324-3p and miR-146b-5p were demonstrated to functionally regulate the expression of IL23R and IL12Rβ1 proteins in CLL cells, respectively. Within lymphoid tissues, in situ, CLL clones expressing IL23R side chain also showed expression of IL12Rß1, which varied according to the density of CD40L-expressing bystander elements suggesting a microenvironment-driven regulation of the IL-23R complex. To functionally test this hypothesis, CLL cells were co-cultured in the presence of NIH-3T3 transduced with CD40L or with AAT cells. A significant up-regulation was observed for both the IL12Rß1 and IL23R side chains, suggesting the environment co-stimulation as a mechanism of IL-23R complex regulation. Consistently, the IL-23R complex was upmodulated in CLL cells expressing IL-23R but not IL12Rß1, upon xenograft with autologous T cells into NOD-Scid mice. We then investigated the effect of IL-23R engagement by IL-23 in CLL cells and found that IL-23R activity correlated with CLL cell proliferation and survival in vitro via STAT3 phosphorylation. The trophic nature of IL-23-mediated stimuli over CLL cells was further demonstrated in vivo through the adoption of an anti-IL23p19 monoclonal antibody for clinical use, which proved to be effective in eradicating the xenografted CLL clone in the infiltrated tissues (spleen, liver and BM) by inhibiting proliferation and inducing apoptosis. Noteworthy, the therapeutic effect of IL-23 antagonism was demonstrated by histopathology, flow cytometry and BCR clonality.Conclusions: Overall, we demonstrated that IL-23/IL-23R axis is a novel microenvironment-regulated determinant in CLL pathobiology representing a strong prospect in disease prognostication and treatment. DisclosuresNo relevant conflicts of interest to declare.

MiR-155 Amplifies AKT and NFkB Signaling By Targeting Multiple Regulators of BCR Signal in DLBCL

IntroductionThe B-cell receptor (BCR)- activated PI3K pathway plays a critical pro-survival function in normal B-lymphocytes and certain B-cell malignancies. Multiple mechanisms are involved in triggering and modulation of the BCR signal amplitude. MiR-155 has emerged as a positive regulator of PI3K signaling in multiple malignancies, including DLBCL, through targeting negative modulator of this pathway, SHIP-1. In the present study we have searched for new targets of miR-155, which might play a role in the deregulation of AKT and NFkB signaling in DLBCL.MethodsMiR-155 target prediction was performed with PicTar, Miranda and TagetScan algorithms. Predicted miR-155 targets were validated with 3'UTR luciferase reporter assays in HEK293 cells. MiR-155 expression was modulated through transfection with miR-155 mimic or miR-155 inhibitor. The consequences of the miR-155 perturbations were assessed in DLBCL cell lines by proliferation assays (MTS) and immunoblotting with antibodies against predicted miR-155 targets and p-AKT. The DEPTOR silencing in DLBCL cells was achieved with retroviral shRNA vector and its consequences were assessed using proliferation assays and immunoblotting. DEPTOR mRNA expression and survival of DLBCL patients was determined using publicly available microarray data (Lenz et al, 2008, GEO accession GSE10846).ResultsUsing miRNA target finding algorithms, we identified miR-155-matching sequences in 3'UTRs of two genes involved in SYK/PI3K/AKT pathway regulation: c-CBL (SYK ubiquitin E3 ligase) and DEPTOR (an mTOR phosphatase). MiR-155 suppressed the luciferase activities of vectors containing 3'UTR fragments from SHIP-1, c-CBL and DEPTOR genes with wild-type, but not mutant miR-155 seed sequence. To establish a link between miR-155 and c-CBL or DEPTOR, DLBCL cell lines were transfected with a control non-targeting miR or miR-155 mimic. The control miR did not affect the protein level of SHIP-1, c-CBL or DEPTOR, whereas introduction of miR-155 resulted in a decrease in expression of these proteins. The repression of miR-155 target genes was accompanied by increased phosphorylation of AKT. As expected, neutralization of endogenous miR-155 in U2932 cell line by anti-miR-155 exhibited opposite effects. In addition to modulation of SYK/PI3K/AKT pathway, miR-155 has been reported to modulate NFĸB activity. To determine whether miR-155 affects NFĸB in DLBCL, we assessed transcript abundance of the genes regulated by NFĸB (CD40, BFL-1, RelB, IĸBα, A20, MIR155HG) in U2932 cell line with reduced miR-155 level. Repression of endogenous miR-155 in these cells led to marked downregulation of NFĸB-controlled genes, indicating that miR-155 amplifies NFĸB signaling in this DLBCL cell line. Consistent with this, anti-miR-155 sensitized U2932 cells to ibrutinib.Since the function of a newly identified miR-155 target, the mTOR phosphatase DEPTOR, in DLBCL has not been elucidated, we silenced the expression of this protein with shRNA. Attenuated protein level of DEPTOR enhanced activity of AKT and promoted proliferation of SU-DHL4 cell line. To determine whether expression level of DEPTOR correlates with patient survival, we analyzed data from gene expression studies. Higher DEPTOR mRNA level in primary DLBCL biopsies was associated with longer overall survival (log rank test, p=0.018). Collectively, these data suggest that DEPTOR plays a tumor suppressor function in DLBCL.ConclusionsOur data underscore the role of miR-155 in the regulation of AKT and NFkB prosurvival signaling in DLBCL. MiR-155 regulates AKT signaling not only by decreasing expression of SHIP-1, but also by modulating the abundance of c-CBL and DEPTOR. DEPTOR modulates AKT activity and its silencing promotes proliferation of DLBCL cells, suggesting that DEPTOR functions as a tumor suppressor in DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Abstract 12208: Microrna-181b Inhibits Thrombin-mediated Activation of Endothelial Cells and Arterial Thrombosis by Targeting Card10 (Best of Basic Science Abstract)

Objective: Thrombogenic and inflammatory mediators such as thrombin induce NF-kB-mediated EC activation and dysfunction, which contribute to the pathogenesis of arterial thrombosis. The role of the anti-inflammatory microRNA-181b (miR-181b) on thrombosis remains unknown. Methods and Results: It has been demonstrated that miR-181b inhibits downstream NF-kB signaling in response to TNF-a. Herein, we reveal that miR-181b can inhibit upstream NF-kB signaling uniquely in response to thrombin. Overexpression of miR-181b inhibited thrombin-induced activation of NF-kB signaling demonstrated by the reduction of phospho-IKK-b, -IkB-a, and p65 nuclear translocation in ECs. MiR-181b also reduced the expression of NF-kB target genes VCAM-1, ICAM-1, E-selectin, PAI-1, and tissue factor. Mechanistically, miR-181b targets Card10, an adaptor protein that participates in the activation of the IKK complex in response to signals transduced from G-protein coupled receptors such as PAR-1. MiR-181b reduced the expression of Card10 mRNA and protein, but not PAR-1. In addition, miR-181b inhibited Card10 3’-UTR luciferase reporter activity, and Argonaute-2 miRNP-IP studies revealed 4-fold enrichment of Card10 mRNA in the RNA-induced silencing complex in the presence of miR-181b, indicating that Card10 is a bona fide direct target. SiRNA-mediated knockdown of Card10 expression phenocopied miR-181b’s effects on NF-kB signaling and targets. Interestingly, knockdown of Card10 does not affect TNF-a-induced activation of NF-kB signaling and target gene expression, suggesting stimulus-specific regulation of NF-kB signaling and endothelial responses by miR-181b in ECs. Finally, in response to photochemical injury-induced arterial thrombosis, systemic delivery of miR-181b reduced thrombus formation by 73% in carotid arteries, and prolonged the time to occlusion by 1.6-fold. Conclusions: MiR-181b is an important regulator of thrombin-induced EC activation and arterial thrombosis. These studies highlight the relevance of miRNA-dependent targets in response to ligand-specific signaling in ECs. Delivery of miR-181b or Card10 inhibition may represent a new therapeutic approach to reduce arterial thrombosis by improving vascular EC function.

MiR-27 impairs the adipogenic lineage commitment via targeting lysyl oxidase.

The recruitment and commitment of mesenchymal stem cells and their terminal differentiation into adipocytes are the main pathways for increasing adipocyte cell numbers during obesity. Our previous studies have shown that lysyl oxidase (Lox) is upregulated and functions as an essential factor during bone morphogenetic protein 4 (BMP4) -induced C3H10T1/2 cell adipocytic lineage commitment. However, the mechanism of Lox regulation during adipogenic lineage commitment has remained largely unestablished. Samples of adipose tissue from humans with different BMI and C57BL/6 mice with a high-fat diet were used to compare microRNA-27 (miR-27) expression level associated with obesity. Taqman assays were used for miR-27 expression detection and Oil Red O staining for adipogenesis analysis. A negative correlation was identified between Lox expression level and miR-27 expression in both BMP4-treated C3H10T1/2 cells and human subcutaneous adipose tissues. A Lox 3' UTR luciferase reporter assay showed that miR-27 directly targeted Lox. Furthermore, overexpression of miR-27 impaired BMP4-induced upregulation of Lox and adipocytic commitment, which could be rescued by overexpression of mature Lox. Conversely, miR-27 inhibition by specific inhibitors increased Lox expression and adipocytic commitment. Taken together, these results suggest a novel role for miR-27 in repressing adipogenic lineage commitment by targeting Lox.

Mmu-miR-351 attenuates the survival of cardiac arterial endothelial cells through targeting STAT3 in the atherosclerotic mice

The signal transducer and activator of transcription 3 (STAT3) signaling pathway was involved in regulation of endothelial cell survival/apoptosis and was regarded as a target for prevention of atherosclerosis or other cardiovascular diseases. Factors, regulating STAT3 expression and activity, have aroused a wide range of interest, such as miRNAs or transcription factors. The aim of this study is to explore the role of miR-351, a miRNA found not long before, in the regulation of STAT3 expression and endothelial cell survival in the model mice with atherosclerosis (AS). Expression of miR-351 in the serum and cardiac arterial endothelial cells of the WT mice and AS mice was detected. Real-time qPCR analysis showed that miR-351 was upregulated in the serum and endothelial cells of the AS mice, displaying an opposite expression pattern with STAT3. To explore the role and mechanism of miR-351 in the endothelial cell survival, the miR-351 mimic was transfected in to the endothelial cells. MTT and Trypan Blue assays showed miR-351 attenuated the survival of endothelial cells. Our results of the TargetScan output and the 3′UTR luciferase reporter assay indicated that STAT3 was target of miR-351. Additionally, miR-351 resisted the elevation of STAT3 protein level and promotion of endothelial cell survival caused by SD19. Finally, our in vitro angiogenesis assay revealed that miR-351 suppressed angiogenesis and resisted the promotion of angiogenesis caused by SD19. In conclusion, miR-351 was upregulated in the atherosclerotic mice. MiR-351 can attenuate the survival of endothelial cells and suppress angiogenesis through targeting STAT3 in vitro.

Detecting new microRNAs in human osteoarthritic chondrocytes identifies miR-3085 as a human, chondrocyte-selective, microRNA

SummaryObjectiveTo use deep sequencing to identify novel microRNAs (miRNAs) in human osteoarthritic cartilage which have a functional role in chondrocyte phenotype or function.DesignA small RNA library was prepared from human osteoarthritic primary chondrocytes using in-house adaptors and analysed by Illumina sequencing. Novel candidate miRNAs were validated by northern blot and qRT-PCR. Expression was measured in cartilage models. Targets of novel candidates were identified by microarray and computational analysis, validated using 3′-UTR-luciferase reporter plasmids. Protein levels were assessed by western blot and functional analysis by cell adhesion.ResultsWe identified 990 known miRNAs and 1621 potential novel miRNAs in human osteoarthritic chondrocytes, 60 of the latter were expressed in all samples assayed. MicroRNA-140-3p was the most highly expressed microRNA in osteoarthritic cartilage. Sixteen novel candidate miRNAs were analysed further, of which six remained after northern blot analysis. Three novel miRNAs were regulated across models of chondrogenesis, chondrocyte differentiation or cartilage injury. One sequence (novel #11), annotated in rodents as microRNA-3085-3p, was preferentially expressed in cartilage, dependent on chondrocyte differentiation and, in man, is located in an intron of the cartilage-expressed gene CRTAC-1. This microRNA was shown to target the ITGA5 gene directly (which encodes integrin alpha5) and inhibited adhesion to fibronectin (dependent on alpha5beta1 integrin).ConclusionDeep sequencing has uncovered many potential microRNA candidates expressed in human cartilage. At least three of these show potential functional interest in cartilage homeostasis and osteoarthritis (OA). Particularly, novel #11 (microRNA-3085-3p) which has been identified for the first time in man.

ANGIOPOIETIN-1 PROMOTES ANGIOGENESIS BY MODULATING MIR-640 EXPRESION IN ENDOTHELIAL CELLS

Aberrant angiogenesis leads to numerous disorders. Angiogenic factors, such as Angiopoietin-1 (Ang-1) are important for angiogenesis. Binding of Ang-1 to its receptor Tie-2 activates mitogen-activated protein kinase (MAPK), phosphinositide 3-kinase (PI3K), and other pathways, which promote EC cell differentiation, proliferation, migration and survival. Ang-1 therapeutic potential has been proved in pre-clinical studies. However its broad spectrum of biological effects limits its use and fine tuning intracellular signals to promote the desired therapeutic actions would be a better approach. Recent studies have revealed important roles for miRNAs in regulating angiogenesis. They can be manipulated in the settings of pathological vascularization and represent a new therapeutic approach. In this study we identified miR-640 as an anti-angiogenic microRNA modulated by Ang-1. To identify new miRNA involved in Ang-1/Tie-2 signaling, HUVECs were stimulated with Ang-1 for 12, 24 and 48hrs and analysed by Illumina miRNA microarray. Array results were confirmed by RT-PCR. The biological function of miR-640 and its target ZFP91 were evaluated by monitoring tube formation by matrigel assay, apoptosis by cell count and cleaved caspase 3, and cell migration by scratch wound assay upon overexpression of the mimic, its inhibitor or siRNA against ZFP91. Target identification was done by 3’UTR luciferase assay, western blot and real-time PCR. Ang-1 treatment lead to dynamic modulation of pre-miR-640 and its mature form. Over expression of miR-640 mimic leads to down regulation of ZFP91 while inhibition of miR-640 leads to increase expression of ZFP91. As expected miR-640 mimic caused a significant loss in luciferase activity while inhibition of miR-640 restored luciferase activity when 3’UTR Luciferase Reporter assay was performed. Mutation of the seed sequence for miR-640 in ZFP91 3’UTR prevented miR-640 mimic reduction of luciferase activity Overexpression of miR-640 mimics leads to an abrogation of Ang-1 induced migration and tubulogenesis but not survival. Similarly transfecting HUVECs with siRNA against ZFP91 blunted Ang-1 induced migration and tubulogenesis. Interestingly, inhibition of endogenous miR-640 leads to a basal increase in migration and tubulogenesis. miR-640 is a new anti-angiogenic microRNA inhibiting Ang-1 induced migration and differentiation. miR-640 exerts its anti-angiogenic properties in part by modulating its target ZFP91. ZFP91 is required for Ang-1 induced migration and differentiation and appears to be a direct target of miR-640. In this study we have extended our current knowledge of pathways regulated by Ang-1 in endothelial cells and identified signaling components regulating specific biological effects.

Integrated stress response is critical for gemcitabine resistance in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with marked chemoresistance and a 5-year survival rate of 7%. The integrated stress response (ISR) is a cytoprotective pathway initiated in response to exposure to various environmental stimuli. We used pancreatic cancer cells (PCCs) that are highly resistant to gemcitabine (Gem) and an orthotopic mouse model to investigate the role of the ISR in Gem chemoresistance. Gem induced eIF2 phosphorylation and downstream transcription factors ATF4 and CHOP in PCCs, and these effects occurred in an eIF2α-S51 phosphorylation-dependent manner as determined using PANC-1 cells, and wild type and S51 mutant mouse embryo fibroblasts. Blocking the ISR pathway in PCCs with the ISR inhibitor ISRIB or siRNA-mediated depletion of ATF4 resulted in enhanced Gem-mediated apoptosis. Polyribosomal profiling revealed that Gem caused repression of global translation and this effect was reversed by ISRIB or by expressing GADD34 to facilitate eIF2 dephosphorylation. Moreover, Gem promoted preferential mRNA translation as determined in a TK-ATF4 5′UTR-Luciferase reporter assay, and this effect was also reversed by ISRIB. RNA-seq analysis revealed that Gem upregulated eIF2 and Nrf2 pathways, and that ISRIB significantly inhibited these pathways. Gem also induced the expression of the antiapoptotic factors Nupr1, BEX2, and Bcl2a1, whereas ISRIB reduced their expression. In an orthotopic tumor model using PANC-1 cells, ISRIB facilitated Gem-mediated increases in PARP cleavage, which occurred in conjunction with decreased tumor size. These findings indicate that Gem chemoresistance is enhanced by activating multiple ISR-dependent pathways, including eIF2, Nrf2, Nupr1, BEX2, and Bcl2A1. It is suggested that targeting the ISR pathway may be an efficient mechanism for enhancing therapeutic responsiveness to Gem in PDAC.

Abstract 450: Suppression of innate and adaptive immune responses in the human lung tumor microenvironment via microRNA-183

Abstract Innate and adaptive immunity plays a critical role in defense against cancer but can be undermined by the tumor microenvironment. To understand the underlying mechanism, we set out to analyze the molecular and cellular basis for natural killer (NK) and CD8 T cell suppression by human lung tumor cells. We found that transforming growth factor-beta (TGFb), abundantly produced by tumor cells, effectively reduced tumoricidal function in activated NK cells and CD8 T cells by specifically targeting a key adaptor protein, DAP12, which is required for surface anchoring of multiple NK receptors, including NKp44. For proof of the TGFb-miR-183-DAP12 circuit, flow cytometric analysis demonstrated that TGFb reduced surface levels of NKp44 on activated primary human NK and CD8 T cells, but western blot analysis demonstrated their intact presence in the cytoplasm. This was due to loss of DAP12, which is critical for surface expression of NKp44 and for downstream signaling to mobilize lytic granules to kill tumor cells. Mechanistically, TGFb readily induced miR-183 expression in both primary human NK cells and T cells which could bind specifically to the 3′UTR of human DAP12 to inhibit transcription and translation. Only miR-183 but not miR-185 or control (CT) transfection repressed luciferase activity of DAP12 3′UTR luciferase reporter constructs transferred into HELA cells. Moreover, we overexpressed lentiviral miR-183 in either primary NK and T cells and found that such expression effectively reduced DAP12 levels in both cell types. Flow cytometric analysis of these same cells indicated that loss of DAP12 was accompanied by loss of NKp44 surface expression and by loss of cytotoxic function against tumor cells. As further proof, we showed that lentiviral anti-sense miR-183 transfection into NK cells and T cells prevented TGFb from suppressing DAP12 expression. Thus, we have uncovered a seminal TGFb-miR-183-DAP12 pathway utilized by tumor cells to silence infiltrating NK and T cells, constituting the first report of miR control of an adaptor protein to control both NK and T cell immune function. Citation Format: Melba Marie Tejera, Melba Marie Tejera, Sarah S. Donatelli, Jun-Min Zhou, Danielle L. Gilvary, Xianghong Chen, Erika A.. Suppression of innate and adaptive immune responses in the human lung tumor microenvironment via microRNA-183. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 450. doi:10.1158/1538-7445.AM2015-450

Abstract 3108: MicroRNA-200B inhibits arsenic-transformed cell migration by targeting protein kinase Cα signaling pathway

Abstract Arsenic is one of the most common environmental pollutants and long term exposure to arsenic is associated with increased risk of multiple types of cancer including lung cancer. Despite extensive studies, the mechanism of arsenic carcinogenicity has not been elucidated. MicroRNAs (miRNAs) are a large class of small non-coding RNAs and have been shown to play important roles in many fundamental processes of cancer. MiRNA-200b (miR-200b) is a member of miR-200 family, which contains 5 members. The miR-200 family has been shown to inhibit tumor cell migration; however, the underlying mechanisms are not clearly defined. We recently found that miR-200 expression is depleted in arsenic-transformed human bronchial epithelial cells, which are highly migratory and invasive. To further study the mechanism by which miR-200 inhibits tumor cell migration, we investigated the effects of stably expressing miR-200b on arsenic-transformed cell migration. We found that stably expressing miR-200b drastically inhibits arsenic-transformed cell migration. Wnt5b and protein kinase Cα (PKCα) are highly expressed in arsenic-transformed cells. Stably expressing miR-200b significantly down-regulated protein levels of both Wnt5b and PKCα. Combining bioinformatics screening and 3′UTR luciferase reporter assays, we identified PKCα but not Wnt5b as a new direct target of miR-200b. We also found that siRNA knocking down Wnt5b greatly reduces PKCα activation while inactivating PKCα drastically decreases Wnt5b level, revealing a positive feedback loop between Wnt5b and PKCα. Knocking down Wnt5b or PKCα phenocopy the effect of stably expressing miR-200b on cell migration. Moreover, enforced expression of PKCα impairs the inhibitory effect of miR-200b on cell migration. These findings not only reveal a novel mechanism showing that miR-200b inhibits tumor cell migration by targeting PKCα and Wnt5b-PKCα positive feedback loop, also suggest that deregulation of miR-200b, Wnt5b and PKCα plays an important role in arsenic carcinogenesis. Citation Format: Zhishan Wang, Brock Humphries, Chengfeng Yang. MicroRNA-200B inhibits arsenic-transformed cell migration by targeting protein kinase Cα signaling pathway. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3108. doi:10.1158/1538-7445.AM2015-3108

Abstract 429: MicroRNA Control of the Pacemaker Channel and Training-induced Bradycardia

Background: Athletes are prone to bradyarrythmias and have a higher-than-normal incidence of pacemaker implantation but the underlying mechanisms are poorly understood. In previous work we demonstrated a training-induced electrical remodelling of the sinoatrial node (SAN), including a down regulation of the key pacemaker channel HCN4 that carries the pacemaker current I f . Here we investigated post-transcriptional regulation of HCN4 by microRNAs (miRNAs) in the trained SN. Methods and Results: Mice subjected to 60-min swimming twice daily for 28 days (TM) were compared to parallel sedentary mice (SM).TM were bradycardic (cycle length in vivo : SM, 81±1.3 ms; TM, 102±3 ms; n=6, p <0.05). The cycle length of the isolated, denervated SAN (intrinsic heart rate) was also prolonged by exercise training (SM, 110±3 ms; TM, 150±5 ms; n=7, p <0.05). Deep sequencing for miRNAs on SAN punch biopsies revealed 10 differentially expressed miRNAs in TM vs SM (FDR adjusted P <0.05); 8 miRNAs were upregulated, 2 were downregulated. These findings were further confirmed by real-time PCR. We used the computation prediction tools Targetscan and Ingenuity Pathway Analysis to identify miR-423-5p and miR-486-3p as putative miRNAs participating in the post-transcriptional repression of HCN4 in the trained SAN. Both miRNAs had conserved seed sequences in the HCN4 3’UTR. To validate whether these miRNAs directly recognise the 3’UTR of HCN4, they were co-transfected with a construct containing HCN4 3’UTR fused downstream to a luciferase coding sequence in H9c2 cells. Transient transfection of precursor miR-423-5p and miR-486-3p decreased HCN4 3’UTR luciferase reporter activity in H9C2 cells by 47% and 22% respectively, compared with unaltered luciferase activity on co-transfection with control miR or miR-27a. Conclusions: This is the first report of miRNA dysregulation in the trained SAN. We describe a novel molecular mechanism whereby an altered miRNA profile in the trained SAN promotes HCN4 downregulation and altered sinus automaticity. Data suggest miR-423-5p as a potential therapeutic target for bradyarrythmias in the athlete.

MicroRNA-106b inhibits osteoclastogenesis and osteolysis by targeting RANKL in giant cell tumor of bone.

Giant cell tumor (GCT) of bone consists of three major cell types: giant cells, monocytic cells, and stromal cells. From microarray analysis, we found that miR-106b was down-regulated in GCT clinical samples and further determined by fluorescence in situ hybridization. In addition, the expression of novel potential target of miR-106b, RANKL, was elevated in GCT along with previously determined targets in other tumors such as IL-8, MMP2 and TWIST. In a RANKL 3'UTR luciferase reporter assays, agomiR-106b repressed the luciferase activity and the effect was eliminated when the targeting site in the reporter was mutated, suggesting a direct regulation of miR-106b on RANKL mRNA. Moreover, overexpression of miR-106b in GCTSCs through TALEN-mediated site-specific knockin clearly inhibited osteoclastogenesis and osteolysis. By grafting the GCT onto the chick CAM, we confirmed the inhibitory effect of miR-106b on RANKL expression and giant cell formation. Furthermore, in an OVX mouse model, silencing of miR-106b increased RANKL protein expression and promoted bone resorption, while up-regulation of miR-106b inhibited bone resorption. These results suggest that miR-106b is a novel suppressor of osteolysis by targeting RANKL and some other cytokines, which indicates that miR-106b may be a potential therapeutic target for the treatment of GCT.

MYB controls erythroid versus megakaryocyte lineage fate decision through the miR-486-3p-mediated downregulation of MAF.

The transcription factor MYB has a key role in hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that MYB controls erythroid versus megakaryocyte lineage decision by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which MYB affects lineage fate decision, we performed the integrative analysis of miRNA and mRNA changes in MYB-silenced human primary CD34+ HPCs. Among the miRNAs with the highest number of predicted targets, we focused our studies on hsa-miR-486-3p by demonstrating that MYB controls miR-486-3p expression through the transactivation of its host gene, ankyrin-1 (ANK1) and that miR-486-3p affects HPCs commitment. Indeed, overexpression and knockdown experiments demonstrated that miR-486-3p supports the erythropoiesis while restraining the megakaryopoiesis. Of note, miR-486-3p also favors granulocyte differentiation while repressing the macrophage differentiation. To shed some light on the molecular mechanisms through which miR-486-3p affects HPCs lineage commitment, we profiled the gene expression changes upon miR-486-3p overexpression in CD34+ cells. Among the genes downregulated in miR-486-3p-overexpressing HPCs and computationally predicted to be miR-486-3p targets, we identified MAF as a miR-486-3p target by 3′UTR luciferase reporter assay. Noteworthy, MAF overexpression was able to partially reverse the effects of miR-486-3p overexpression on erythroid versus megakaryocyte lineage choice. Moreover, the MYB/MAF co-silencing constrained the skewing of erythroid versus megakaryocyte lineage commitment in MYB-silenced CD34+ cells, by restraining the expansion of megakaryocyte lineage while partially rescuing the impairment of erythropoiesis. Therefore, our data collectively demonstrate that MYB favors erythropoiesis and restrains megakaryopoiesis through the transactivation of miR-486-3p expression and the subsequent downregulation of MAF. As a whole, our study uncovers the MYB/miR-486-3p/MAF axis as a new mechanism underlying the MYB-driven control of erythroid versus megakaryocyte lineage fate decision.

ADAM12-L is a direct target of the miR-29 and miR-200 families in breast cancer.

BackgroundADAM12-L and ADAM12-S represent two major splice variants of human metalloproteinase-disintegrin 12 mRNA, which differ in their 3′-untranslated regions (3′UTRs). ADAM12-L, but not ADAM12-S, has prognostic and chemopredictive values in breast cancer. Expression levels of the two ADAM12 splice variants in clinical samples are highly discordant, suggesting post-transcriptional regulation of the ADAM12 gene. The miR-29, miR-30, and miR-200 families have potential target sites in the ADAM12-L 3′UTR and they may negatively regulate ADAM12-L expression.MethodsmiR-29b/c, miR-30b/d, miR-200b/c, or control miRNA mimics were transfected into SUM159PT, BT549, SUM1315MO2, or Hs578T breast cancer cells. ADAM12-L and ADAM12-S mRNA levels were measured by qRT-PCR, and ADAM12-L protein was detected by Western blotting. Direct targeting of the ADAM12-L 3′UTR by miRNAs was tested using an ADAM12-L 3′UTR luciferase reporter. The rate of ADAM12-L translation was evaluated by metabolic labeling of cells with 35S cysteine/methionine. The roles of endogenous miR-29b and miR-200c were tested by transfecting cells with miRNA hairpin inhibitors.ResultsTransfection of miR-29b/c mimics strongly decreased ADAM12-L mRNA levels in SUM159PT and BT549 cells, whereas ADAM12-S levels were not changed. ADAM12-L, but not ADAM12-S, levels were also significantly diminished by miR-200b/c in SUM1315MO2 cells. In Hs578T cells, miR-200b/c mimics impeded translation of ADAM12-L mRNA. Importantly, both miR-29b/c and miR-200b/c strongly decreased steady state levels of ADAM12-L protein in all breast cancer cell lines tested. miR-29b/c and miR-200b/c also significantly decreased the activity of an ADAM12-L 3′UTR reporter, and this effect was abolished when miR-29b/c and miR-200b/c target sequences were mutated. In contrast, miR-30b/d did not elicit consistent and significant effects on ADAM12-L expression. Analysis of a publicly available gene expression dataset for 100 breast tumors revealed a statistically significant negative correlation between ADAM12-L and both miR-29b and miR-200c. Inhibition of endogenous miR-29b and miR-200c in SUM149PT and SUM102PT cells led to increased ADAM12-L expression.ConclusionsThe ADAM12-L 3′UTR is a direct target of miR-29 and miR-200 family members. Since the miR-29 and miR-200 families play important roles in breast cancer progression, these results may help explain the different prognostic and chemopredictive values of ADAM12-L and ADAM12-S in breast cancer.

MiR-106b-5p targets tumor suppressor gene SETD2 to inactive its function in clear cell renal cell carcinoma.

Inactivation of human SET domain containing protein 2 (SETD2) is a common event in clear cell renal cell carcinoma (ccRCC). However, the mechanism underlying loss of SETD2 function, particularly the post-transcriptional regulatory mechanism, still remains unclear. In the present study, we found that SETD2 was downregulated and inversely correlated with high expression of miR-106b-5p in ccRCC tissues and cell lines. Over-expression of miR-106b-5p resulted in the decreased mRNA and protein levels of SETD2 in ccRCC cells. In an SETD2 3'-UTR luciferase reporter system, miR-106b-5p downregulated the luciferase activity, and the effects were abolished by mutating the predicted miR-106b-5p binding site. Moreover, attenuation of miR-106b-5p induced cell cycle arrest at G0/G1 phase, suppressed cell proliferation, enhanced processing of caspase-3, and promoted cell apoptosis in ccRCC cells, whereas these effects were reversed upon knockdown of SETD2. In addition, transfection of miR-106b-5p antagomir resulted in the increased binding of H3K36me3 to the promoter of p53 and enhanced its activity, as well as upregulated the mRNA and protein levels of p53, and the effects were also abolished by cotransfection with si-SETD2. Collectively, our findings extend the knowledge about the regulation of SETD2 at the posttranscriptional level by miRNA and regulatory mechanism downstream of SETD2 in ccRCC.